The proteolipid subunit of the chloroplast adenosine triphosphatase complex. Reconstitution and demonstration of proton-conductive properties.

The dicyclohexylcarbodiimide (DCCD)-binding proteolipid of the chloroplast ATPase complex was solubilized in 1-butanol, isolated by ion exchange chromatography, and reconstituted in a liposomal system. Proton-conducting activity was monitored by fluorometry with 9-aminoacridine as an indicator of delta pH in K+-loaded liposomes suspended in a K+-free medium. Addition of valinomycin served to create a membrane potential. Proton mediation was further followed potentiometrically with a pH electrode. Reconstituted chloroplast DCCD-binding proteolipid rapidly catalyzed passive proton movement as measured by the quenching of 9-aminoacridine fluorescence upon addition of valinomycin to K+-loaded vesicles. Proton translocation was inhibited to approximately 80% by hydrophobic dicyclohexylcarbodiimide but not its water-soluble analog (1-ethyl-3[3-dimethylaminopropyl]carbodiimide). Maximal inhibition by DCCD occurred after 60 min of incubation with 20 to 40 nmol of DCCD/20 nmol of proteolipid/10 mumol of lipid. The velocity of proton conduction increased when the external proton concentration in the medium was increased. Similar data were obtained using a pH electrode to follow proton movement. Subjection of proteolipid liposomes to tetranitromethane, resulting in nitration of tyrosine, markedly reduced proton conduction (62% inhibition). In a similar fashion preincubation of reconstituted vesicles with hydrophobic phenylisothiocyanate at neutral pH resulted in a decreased rate of proton movement (50% inhibition) while hydrophilic p-sulfophenylisothiocyanate appeared to have no effect.